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...and a screening approach, the Jennifer Doudna lab identified three different Cas12a inhibitors. Some of ...hisashii, BhCas12b that is functional at 37 °C but preferentially nicks the non-target DNA strand. The lab then... of stable genomic integration and is easily transferred via conjugation.   Read the paper in Nature... Jennifer Tsang...

...sharing even broader and began working with tech transfer offices to make reagents available to scientists... Proteins For Molecular Biology Experiments by Jennifer Tsang Chromoproteins are a subset of the fluorescent.... 7. 25 Work From Home Tips for Scientists by Jennifer Tsang These tips were written early on in the...Five DNA Quantification Methods to Consider by Jennifer Tsang The nanodrop vs. spec debate dominates ... Jennifer Tsang...

...dependent cell lines and any mutations that that confer proliferation advantages will be revealed in the absence... a given mutation will have a different phenotypic effect in a different system. These plasmids can also...well-studied candidates. The possibilities are vast. References Ng, Patrick Kwok-Shing, et al. "Systematic functional...

...use in this service.  At launch, we will offer five different serotypes for packaging: AAV1, AAV2, AAV5...the Addgene repository and their choice of five different serotypes.   Figure 1: The process of...that this ordering process will look slightly different from our standard process. We’ll walk you through... Preps will be available for request in three different quantities: 10x 100 µL (1 mL), 25x 100 µL (2.5...

...neuronal populations to consider. To tease apart how different neurons impact a certain output, neuroscientists... populations using viral strategies Combining different approaches can increase the specificity of cell...optogenetic payload in neurons that project to different regions of the brain. A more locally defined set...in their cell bodies. Optical stimulation in a different brain area can then reveal the cells bodies of...share experimental design and data. With these different approaches to open science, we aim to accelerate...

...NmCas9 and St1Cas9. Each ortholog was fused to a different fluorescent protein to create three colors. The...specificity of these orthologs is key: due to differences in the PAM sequences required by each ortholog...specific for telomeric sequences and showed that different fluorescently labeled dCas9 isoforms are efficiently...target sequence. They were also able to label two different pairs of chromosomes using gRNAs specific to sequences...Addgene are excited to see what’s coming next! References Multicolor CRISPR labeling of chromosomal loci...

...the efficiency of Cre recombination varies for different constructs and cell types. Second, Cre may induce...from the standard UAS/GAL4 system is toxic to proliferating cells. A Cre-estrogen receptor ligand binding...enhance genome editing capabilities in Drosophila. References: Multiple new site-specific recombinases for ...for use in manipulating animal genomes. Nern A, Pfeiffer BD, Svoboda K, Rubin GM. Proc Natl Acad Sci USA...PubMed. Reduction of Cre recombinase toxicity in proliferating Drosophila cells by estrogen-dependent activity...

...Dr. Jennifer Doudna and Dr. Emmanuelle Charpentier. I was joined by Addgene Scientist Matt Ferenc (one...especially excited to attend this event because Jennifer did her graduate work one floor above mine in ...system Drs. Dounda and Charpentier met at a 2011 conference and decided to collaborate on what was a “curiosity... science to the non-scientists in the room. He offered the great analogy of comparing the CRISPR/Cas9 ...

...online on Addgene's CRISPR Library Reference page.    References: Genetic Screens in Human Cells Using...GeCKO library, each library has been enriched for different gene targets. These pooled libraries include enrichment...lentiviral libraries and their demonstrated application offer proof-of-principle that CRISPR-Cas9 can be used ...any sequence in the genome. Tips and tricks Wang offers this tip: “It's quite important, at every step ...

...at how variable Cas9-expression was across the different cells (Figure 3).   Figure 2: Cas9 expression...publication. Pro-Tip: 1. Use multiple gRNAs to target different regions of your gene. I used existing BRAF gRNA...gRNA viruses. There are a few reasons to target different regions of the gene with multiple gRNAs, but the...comparing the phenotypes of multiple gRNAs. Targeting different regions of the gene is also useful in case the...that would co-migrate on a gel) instead of two different sized digestion products (which could be easier...your way to making new scientific observations. References 1. Rago C, Vogelstein B, Bunz F. Genetic knockouts...

... just find one that naturally occurs? This is different than the engineered approach used to make eSpCas9...SpCas9 or GeoCas9 RNPs were incubated in reaction buffer at temperatures ranging from 25-80 °C. Activity...incubated in human plasma diluted with reaction buffer for 8 hours at either 37 °C or 4 °C. Substrate ...gel. SpCas9 is stable when incubated in reaction buffer alone, but has limited activity when incubated ...are available from Addgene. Happy CRISPRing! References Harrington, Lucas, et al. "A thermostable Cas9...

...fundamentals. Our mission is to curate a one-stop reference guide for plasmids, so that you can spend less...their artificial nature, lab plasmids are commonly referred to as “vectors” or “constructs.” To insert a gene...Green Fluorescent Protein (GFP) Plasmids 101: Luciferase Monitor the level of a chemical in a particular...plasmid use in the lab. Addgene’s Molecular Biology Reference includes information about molecular cloning, ...a plasmid vector, molecular biology tools and references, and how to maintain your plasmid stocks. The...

...antibodies, and more!   Resources and References   References Coligan, J. E., Dunn, B. M., Speicher, ...blot and immunofluorescence. GST  Glutathione S-Transferase (GST) is a 26 kDa tag which can be isolated with...Nonetheless, this tag can be purified with several different resin types and is a fairly small tag that is ...  Affinity tags vs. other tags There are many different types of tags, including epitope tags (FLAG, Myc...

...tools for your next experiment! References and Resources References Wood, V., et al., Hidden in plain...an endogenous gene. Each trap uses a slightly different approach (described in detail below), but fundamentally...kidneys at a specific developmental stage you could infer that the gene which GFP was monitoring was being... these circumstances, trap methods may be the preferred screening option. Promoter traps Promoter trap...

...specific compounds, links to publication references, and cross-references with cell line catalogs and collections...be adjusted based on query, reference, and shared allele search preferences to help you identify proximal...to learn more, check out the Cellosaurus FAQ! References Lorsch JR, Collins FS, Lippincott-Schwartz J....

... with a six-year-old boy named AJ Gonzales who suffers from Fibrodysplasia ossificans progressiva (AKA...first met AJ and his family, he did not seem much different than my second son of similar age. When he came...therapeutic antibody that binds Activin A and interferes with inappropriate activin-ACVR1 interactions...superpower for those running on behalf of rare disease sufferers like AJ. As the Blue Lantern Oath says: "In fearful...running on behalf of AJ Gonzales, a young boy suffering from FOP, raising money for NORD for the past ...

...encompasses millions of choices including pharma, tech transfer, management consulting, science communication,...of today’s postdoctoral training can directly interfere with a seamless jump into industry.” In his article...interact with scientists just like you What is different about academia? Publishing in peer reviewed journals...Constant worry about grants and renewals What is different about non-academia? Scientific publications are...where you want to go will help you focus on the transferable skills that you will need to get there. Additional...

...work demonstrates that different repair template strategies activate different HDR sub-pathways like synthesis-dependent...live updates! My most popular tweet from the conference is shown above. The blog post I mentioned, John...post-CRISPR cleavage When I first looked at the conference agenda, I was surprised to see that it would ...Having had time to digest my notes, since the conference, I think I may have finally grasped just how ...

...due to genetic drift and the use of genes from different microbial strains. It proved to be impractical... least 96% sequence identity when detecting a reference feature. For a coding sequence feature that may... could miss a common feature if the sequence differences exceed the threshold. This issue is addressed...often come in closely related versions that have different properties. To prevent misidentification, we augment...way for SnapGene users to apply features from a reference strain to newly sequenced strains through our ...

...Request service, which offers over 3,000 eligible transfer plasmids and five different serotypes. Not sure...pAAV_hSyn1-SIO-stGtACR2-FusionRed AAV5 Optogenetics Ofer Yizhar New serotype pAAV-CaMKIIa-ChRmine-oScarlet-KV...pAAV-CaMKIIa(0.4)-eOPN3-mScarlet-WPRE AAVrg Optogenetics Ofer Yizhar New serotype pAAV-syn-Flpo AAVrg Recombinase...

...transductions. If you’ve ever wondered how they are different from or similar to the other Ts, we have your ...transduction?  Transduction is the process of transferring foreign nucleic acid into a cell by a viral ...controlled lab usage. Using viral transductions can offer benefits such as tissue-specificity, high efficiency...that you can swap out your virus type to achieve different biological outcomes. For example, some viruses...target cells or organism, some primary cells and differentiated cell types are picky when it comes to transfection...

...cell-free transcription/translation systems (also referred to as TX-TL systems) have been gaining popularity...initial system by using a stronger promoter and different biochemical secondary energy sources to increase...XMAS-TREE. a) Cells are transfected with three different vectors and flow cytometry is used to sort populations...XMAS-TREE-based methods at various genomic loci in four different categories.     Brookhouser, N., et al. BMC...10.1186/s12915-020-00929-7    The CRISPR corner By Jennifer Tsang New CRISPR plasmids are always being added...

...Khammash lab can also be found here.   References and Resources References Romano E, Baumschlager A, Akmeriç...functional BLADE variants, too. The team tested different linkers between VVD and the DNA-binding domain...the Molecular Biology Reference Download Addgene's Plasmids 101 eBook ... Jennifer Tsang...

...antibodies for tagging purposes. Different antibodies bind different protein sequences called epitopes...infamous frankenstein, was constructed by grafting different parts together. By combining two optimized elements...living cells. To make these scaffolds specific to different targets, the scientists fused them to sequences...to build a more robust set of frankenbodies. References Ewert, S., Honegger, A., & Pluckthun, A. (2004...

...However, careers outside of the lab highlight “transferable” skills such as leadership, management, and ... and trends “Never Leave an Empty Seat” at conferences and seminars, as this is a valuable time to network...particular field of study Assess your interest in a different field Outreach activities (e.g. Volunteer...Leadership Networking University technology transfer office positions   Market analyses Patent...Marketing Communication Business models Conference or club organizer Management Leadership...

...numerous cell lines can be generated, each with a different tagged protein of interest, and a universal all-purpose.... Current methods of epitope tagging, however, suffer from many pitfalls: they require construction of... achieve 5-30% tag knock-in efficiency across different stem cell types. This has facilitated easy generation...format and tested parallel tag knock-in for 96 different genes in a single go (Figure 3). We were able ...optimization includes selection of transfection buffer and electroporation pulse program to achieve high...seen daydreaming about sunny days in the UK.   References 1. Bressan RB, Dewari PS, Kalantzaki M, Gangoso...

... Gal4 lines and study gene expression in many different tissue types! Although putting a gene of interest... overexpressed in several cell types by using different Gal4 drivers. It is much easier to mate flies ...one gene in one cell type and another gene in a different cell type (6–8). Although it’s often very easy...the meantime, be sure to browse the publication references and web links from my first post and start designing...jonchowphd but he still reads “#” as a “pound symbol”. References 1. A. H. Brand, N. Perrimon, Targeted gene expression...

...heart disease, and type 2 diabetes, across four different types of human cells, including T cells and hematopoietic... interest with only minimal genetic payloads. Different from existing multiplex array architectures, including...when repetitive tRNA sequences must be avoided, different tRNAs with optimized 5’ leader sequences can be... lab of Xue Sherry Gao at Rice University.    References and Resources More resources on the Addgene blog...Adding Precision and Flexibility to CRISPR Editing References Yuan Q, Gao X (2022) Multiplex base- and prime-editing...

...seeing adoption of the technology in different labs for different applications.” Li’s team noted that ...cells of patients with cystic fibrosis (8).   The difference with CRISPR/Cas9 in comparison to other genome...” said the University of California Berkeley’s Jennifer Doudna. “They are the first direct demonstration...possible through the use of nanoparticles. [6].    References 1. Zhang B. 2021. “CRISPR/Cas gene therapy.” ...

...patterns of different AAV serotypes. To help make this easier for scientists, we now offer trial size ...excited to offer these useful tools to the community. Among other new biosensor tools, we now offer the jGCaMP7c...control of specific neuronal populations. We now offer a Flp-expressing AAV, as well as a Cre-dependent...

...identifiers (PIDs), a long-lasting way to identify and reference documents, files, or physical reagents. What ...and exist online indefinitely. PIDs historically referred to objects that exist online, but this approach...you, we’ll go through examples to highlight the difference between them using a plasmid available through...the lenti-Cas9-VQR plasmid page. Despite their differences, both of these PID formats for lenti-Cas9-VQR...managed by Addgene. How to cite options using the different PID formats The instructions for how to cite any...

... that affect Th2 differentiation and activation, using expression of differentiation factors as the screen...ApoB-NanoLuc reporter by inserting the NanoLuc luciferase downstream of the apoB gene. Using this reporter... imaging can be performed in intact animals and offers many advantages over traditional measurements such...Gottschalk group hypothesizes that this may be due to different lipid compositions of C. elegans membranes, which...

...for creative interaction During Zoom meetings, different tools can be used to promote creative interaction...as a picture. I believe that showing something different from time to time breaks up the monologue a little...papers will be written by several authors from different institutes, disciplines, and cultural backgrounds...and only general chatter will be allowed with a different host each time that moderates conversations. This...communities. You can follow him on twitter @salmon_phd References Frassl MA, Hamilton DP, Denfeld BA, de Eyto E...

...events for one organization or go to the same conferences/meetings over and over to get to know the regulars...people by discussing non-work topics and interests. Offer help when you can, instead of just asking for favors...starfish shape. You can ask me about the different arms of this starfish sometime, but as of yesterday...come across your contacts over the years in many different ways. This leads me to my last tip: It’s usually...Browse other blog posts on networking Attending a conference soon? Get tips here Learn about informational...

... the library are differently sized, smaller plasmids may be amplified preferentially, skewing the composition...some of Addgene’s most exciting and versatile offerings! We recently re-amplified our distribution stock...successful amplification? To provide a point of reference, Addgene commonly prepares NGS samples using both...overrepresented plasmids (plasmids that were amplified preferentially). You can also rank the plasmids by abundance...

... will be depositing multiple plasmids and would prefer to send us your plasmid information in spreadsheet...plasmids all built with the same backbone and only differing in the gene/insert. Pooled libraries are normally...click the "Save" button and jump around to the different pages by clicking on the appropriate boxes at ...at the bottom of the page before moving onto a different page so that any data you enter is not lost.   ...to the backbone, and the vector type. Pro Tip! Reference the Addgene ID in the "Backbone Manufacturer" ...concentration of 0.1–1 µg/µL (or bacterial streaks, if you prefer). If you wish to edit your plasmid data after submission...

...organizers. My overall impression from this conference was that Cancer Researchers and Immunologists...Here I aim to compile a few highlights from the conference which reflect how cancer/immunology researchers...multi-pronged attack. Inflammation and tumorigenesis The conference opened with a very interesting presentation by...our Fluorescent Protein Guide. Throughout the conference aspirin and the use of COX inhibitors was discussed...about our free deposit and distribution service! References: 1. Pietrocola, Federico, et al. "Caloric restriction...

...mutation of your favourite gene, there are several different methods available. Thankfully, there are many ...mutations. The assays you will use aren’t that much different from conventional duplexed primer probe assays...primer to amplify the target locus, as well as a reference probe designed to bind at an unedited site distal...are completely “blind” to this very important difference (Figure 3). If you are interested in harnessing...biology. Follow him on Twitter @AnotherLabRat.     References 1. Miyaoka, Y. et al. Isolation of single-base...

...AAV8) enable different patterns of transduction and transgene expression across different cell types (...world. We began with only a few inventory items offered domestically, but by the end of 2016, we expanded...visualizing hM4D(Gi)-mCherry in fixed tissue. References 1. Aschauer DF, Kreuz S, Rumpel S. Analysis of...Kilic E, Bähr M. Human synapsin 1 gene promoter confers highly neuron-specific long-term transgene expression...

...Pro tip! If your samples and control underwent different treatment processes, you may see a shift in the...presence or absence, the plots can look quite different. You might have high expression, low expression... the mean fluorescence intensity (MFI) of the different populations. MFI is the average brightness from...plot. However, the histogram plots look a bit different than they do for yes-no readouts.      ...above is for a smeary expression and is quite different from the yes-no histogram plot shown previously...